Luminescent chemistries have become increasingly important as an effective means of achieving high bioassay sensitivity without the use of radioisotopic labels. The most efficient luminescent reaction known is catalyzed by beetle luciferases. Thus, luciferases are commercially valuable in a wide range of applications, including basic and clinical research, clinical and industrial diagnostics, and drug discovery. However, present technology is dominated by only the common firefly luciferase, which lacks sufficient physical stability for many commercial opportunities. Molecular genetics technology provides us access to the widely diverse group of beetle luciferases, and enables us to manipulate their structures. Building on previous research in our laboratory, the Phase II research will extend our analysis of beetle luciferases to new groups found within juvenile forms of luminous beetles. The genes encoding these juvenile-form luciferases were cloned in Phase l. The enzymes will be characterized in Phase Il, and we will choose those most commercially suited for final optimization. This will be achieved using a mutagenesis strategy based on general principles of enzyme structure and natural evolution. In this strategy, process efficiency will be attained through population genetics and multiple quantitative selection criteria of enzyme performance. Automation will be used to maintain high process throughput. PROPOSED COMMERCIAL APPLICATION: The luciferases to be developed in Phase II will provide the basis for many new product areas. These will be developed in Phase III, particularly for biomedical research, pharmaceutical research and development, and clinical and industrial diagnostics. The estimated value of each of these market areas is several million dollars annually. These new reporter technologies that increase both assay throughput and accuracy are essential in small molecular screens for new drug discovery and in development of new diagnostics systems.